1. Field of the Invention
The present invention relates to a separation analysis system for analysis of an analyte containing many kinds of components. More particularly, this invention relates to a liquid chromatograph/mass spectrometry system, an analyzing method and equipment which are used for analysis of organism-related substances such as proteins, peptides, oligosaccharides, and metabolites.
2. Description of the Related Art
In recent years, a proteome analysis, a glycome analysis, and a metabolome analysis which allow comprehensive analysis of organism-related substances, such as proteins, oligosaccharides, metabolites which are contained in an organism texture and a body fluid, are being recognized to be effectively strategic mainly by those in the fields of biochemistry, innovative drug development, and food. For the analysis of them, the development of a high throughput analysis technology mainly using the mass spectrometry is emphasized.
In the mass spectrometry, a technology for high efficiently ionizing organism-derived molecules having high molecular weight has been advanced. The mass/charge ratio m/z of the ionized molecules can be measured using a mass spectrometer.
Molecules constructing an ion contain isotopes at a constant rate, whereby a series of ions containing isotopes is observed at the same time. The interval on the m/z axis is 1/z so that the charge of an ion can be judged. The mass m of an ion is calculated from the m/z and z values. Furthermore, the use of an ion trap method allows one of ions observed in the first-time mass spectrum (hereinafter referred to as MS1) to be selectively left. This ion serves as a parent ion to be fragmented using a CID (Collision Induced Dissociation), an IRMPD (InfraRed MultiPhoton Dissociation), or an ECD (Electron Capture Dissociation), whereby the spectrum of the fragment ion can be obtained (hereinafter referred to as MS2; fragment ion is also called a product ion). One of the obtained fragments can further be fragmented (MS3, MS4 and so on; hereinafter, a measurement is described as MSn, where n≧2). The sort of a fragmenting method and a fragmenting energy can be controlled to dissociate the particular kind of the intramolecular binding during the process of fragmentation. Information on the molecular structure of the parent ion can be obtained from the fragment ion resulted from fragmentation. When information is insufficient, continuation of the MSn measurements provides further information.
For example, in the analysis of the peptide which is a polymer of several tens of molecules of amino acids, fragmentation is performed such as to break the binding between amino acids, and the fragments having a difference in mass that matches the mass of one of the twenty kinds of amino acids are sequentially found from many kinds of fragments generated by the breaking of any binding, whereby an amino acid sequence of the peptide can be read sequentially from the edge. At this time, the information for identifying substances can be efficiently obtained by automatically reading the amino acid sequence from the peptide fragments observed in the spectrum before the obtaining of the next spectrum to select the most appropriate ion as a parent ion for the next fragmentation. This method can be said to be particularly useful technology when carrying out LC/MS (liquid chromatography/mass spectrometry) measurement because the time width in which a certain component is eluted from an LC is limited (refer to Japanese Unexamined Application Publication No. 2004-257922).
For example, in the analysis of oligosaccharide, fragmentation is performed such as to break the binding between monosaccharides or the cyclic structure of the monosaccharide to observe a fragment specific to a certain oligosaccharide, the structural information of which has been registered in a data base(hereinafter described as “known”), whereby the oligosaccharide to be analyzed can be identified in some cases (refer to Journal of the American Society for Mass Spectrometry (2002) p. 1138-1148).
A sugar includes many stereo isomers having the same mass. There also exist two kinds α and β of cyclization forms sterically different from each other. Furthermore, the oligosaccharide which is formed by chain polymerization of monosaccharides also includes ones having a branched structure. The oligosaccharides which have the same mass but have many different structures are therefore likely to be available. Therefore, it is difficult to uniquely determine the structure of the oligosaccharide, the structural information of which has not been registered in the data base (hereinafter described as “unknown”), by means of the mass spectrometry.
Therefore, with respect to multiple kinds of known substances, the mass spectra of the fragments obtained under certain ionization and fragmentation conditions are previously prepared, and the analyte is then measured under the same conditions as above to obtain a mass spectrum. A spectrum matching technique where the obtained spectrum pattern is compared with that of the previously prepared spectrum can subsequently be performed to judge whether the analyte is the same as a known substance.
The data base of the substances which tend to desorb during fragmentation is previously established. Then, the data base is searched to determine the attribution of fragment ion, whereby the candidate of a parent ion can be determined by finding the combination of the fragment ion with a desorbed substance (refer to Japanese Unexamined Application Publication No. Hei8-124519).